Application of tumor model multicellular spheroids for combination therapy of siRNA targeting HIF-VEGF and radication

目的：缺氧诱导因子（HIP）和血管内皮生长因子（VEGF）的表达在耐辐射缺氧肿瘤细胞中升高。因此，我们假设抑制HIF和VEGF功能可能会增强辐射的细胞毒性作用。为了阐明这一点，我们使用小干扰RNA （siRNA）来抑制这些基因功能和多细胞球体作为三维肿瘤模型。材料和方法：用HIF/VEGF siRNA和/或辐射处理单层和球状人肺癌细胞SQS。采用定量RT-PCR和western blotting分析基因表达。通过菌落形成试验确定细胞毒性。结果：与单层细胞相比，球状细胞中HIF和VEGF的表达水平均稳定上调。用HIF或VEGF siRNA处理在单层细胞和球状细胞中均显示出这些mRNA表达的抑制作用。低氧条件明显诱导单层细胞和球形细胞的抗辐射能力。在低氧条件下，HIP siRNA增强了单层细胞的辐射效应，而VEGF siRNA不增强。HIF或VEGF siRNA在常氧或缺氧条件下均未增强球体的辐射效应。结论：球体可用于评价肿瘤对siRNA下调基因表达的反应。低氧条件下，HIF siRNA对单层细胞表现出辐射增强作用，诱导细胞耐辐射。

Purpose: Expression of hypoxia-inducible factor (HIP) and vascular endothelial growth factor (VEGF) elevated in hypoxic tumor cells that show resistant to radiation. Therefore, we hypothesized inhibiting HIF and VEGF function may enhance the cytotoxic effects of radiation. To clarify this point, we use small interference RNA (siRNA) to inhibit those gene function and multicellular spheroid as a 3D tumor model.Materials and methods : Monolayer and spheroids of human lung carcinoma cell SQS, were treated with HIF/VEGF siRNA and/or radiation. Quantitative RT-PCR and western blotting were done to analyze gene expression. Cell toxicity was qualified by colony formation assay.Results : In spheroid, the expression levels of both HIF and VEGF were stably up-regulated compared with monolayer cells. Treatment with HIF or VEGF siRNA displayed inhibition of those mRNA expressions in both monolayer cells and spheroid. Hypoxic condition apparently induced radiation resistance of monolayer cells and spheroid. HIP siRNA but not VEGF siRNA enhanced radiation effect on monolayer cell under hypoxic condition. Either of HIF or VEGF siRNA did not enhance radiation effect on spheroid either normoxic or hypoxic condition.Conclusion : Spheroids can be applied for evaluating tumor response to down-regulation gene expression by siRNA. HIF siRNA showed radiation enhancement effect on monolayer cells under hypoxic condition in which radiation resistance of cells were induced.